Research reportNeuronal localization of fibroblast growth factor-9 immunoreactivity in human and rat brain
Introduction
Fibroblast growth factor-9 (FGF-9), or glia-activating factor, is a relatively new member of the FGF family that now consists of at least 14 members 25, 34. This factor was originally isolated from the conditioned medium of a human glioblastoma cell line (NMCG-1) as heparin-binding proteins that possess a growth-stimulatory activity on rat primary glial cells 19, 30. Molecular cloning of the cDNA revealed 30% sequence similarity to other members of the FGF family, and the FGF-9 gene is highly conserved between human, rat and mouse 10, 16, 23, 24. The physicochemical properties of FGF-9, such as instability to heat or acid and high affinity for heparin, are similar to those of prototype FGFs (acidic FGF/FGF-1 and basic FGF/FGF-2) [19]. FGF-9 also resembles prototype FGFs in that it has no typical signal sequence in its N-terminus [16]. Prototype FGFs are primarily considered as cell-associated proteins, although an energy-dependent, non-Golgi export pathway has been recently identified for the 18-kD isoform of basic FGF [7]. FGF-9 is somewhat unique among the FGF family in that, despite the lack of a typical signal sequence, it is basically secreted from cells after synthesis like those other members that have typical signal sequences. It has been shown that, when COS cells were transfected with FGF-9 cDNA, the factor was detected exclusively in the culture supernatant and not in cell lysates [16]. FGF-9 is also distinct from prototype FGFs in that it exhibits no mitogenic effect on human umbilical vein endothelial cells, whereas acidic and basic FGFs are well-known for their angiogenic activities [19].
Recent and accumulating findings suggest that members of the FGF family, especially acidic and basic FGFs, are importantly involved in the development and maintenance of the nervous system 5, 20, 28, 31. Although the physiological functions of FGF-9 are still unknown, its close relation to the central nervous system has been implicated by several evidences, not to mention its stimulatory action on cultured glial cells, for which it was originally characterized and named. It has been demonstrated by Northern blot analysis that FGF-9 is expressed in the brain and the kidney of an adult rat [16]. By using a sensitive enzyme immunoassay (EIA), the factor has been detected in the extract of rat cerebellum but not from other organs [13]. Most recently, it has been shown by in situ hybridization that FGF-9 mRNA is preferentially expressed in neurons in the rat brain [27]. To further elucidate the roles of FGF-9 in the central nervous system and considering the secretory nature of this growth factor, it is important to disclose the distribution of the FGF-9 proteins in the normal brain. In this paper, we investigated in detail the localization of FGF-9 proteins in the human and rat brains by using immunohistochemical techniques.
Section snippets
Antibodies
Two different antibodies were used in this study to detect FGF-9. A polyclonal antibody was purified from serum of a rabbit immunized with recombinant human FGF-9 (rhFGF-9 N33) as previously described [14]. Another polyclonal antibody was raised in rabbits against a synthetic peptide corresponding to amino acid residues 87-102 of human FGF-9 [18]. The sequence is unique for FGF-9, but is common between human and rat FGF-9. Both antibodies have been extensively characterized for their
Results
Results obtained from the two different antibodies used to detect FGF-9 were essentially similar in both human and rat. In the human cerebral cortex, neurons of all layers showed strong immunoreactivity for FGF-9 (Fig. 1A). All neurons in the cerebral cortex were uniformly stained in intensity, and the reaction product was localized mainly in the perikaryon and the proximal processes. Neuropil was also weakly stained. Similar neuronal localization of FGF-9 immunoreactivity was observed in the
Discussion
In this paper, we demonstrate that a strong expression of FGF-9 immunoreactivity is mainly localized in neurons throughout the normal brain in both human and rat. In the human brain, strong and uniform immunoreactivity was observed in the neurons of cerebral cortex, hippocampus, substantia nigra, motor nuclei of the brainstem, and Purkinje cell layer. Compared to the human brain, the rat brain showed more variation of the intensity of FGF-9 immunoreactivity in neurons between different
Conclusion
In conclusion, FGF-9 immunoreactivity is localized preferentially in neurons throughout the normal brain in both human and rat. FGF-9 appears to be potentially an important growth factor for the maintenance and development of the central nervous system. We believe that this detailed mapping of the expression of FGF-9 immunoreactivity in the normal brain forms an important basis for the future investigations to elucidate the physiological functions of FGF-9 in the brain.
Acknowledgements
This work was supported in part by research grants from the Ministry of Health and Welfare (to T.T., T.Ko. and K.I.) and the Ministry of Education, Science and Culture (to T.Ki. and K.I.) of Japan.
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